Iron attenuates erythropoietin production by decreasing hypoxia-inducible transcription factor 2α concentrations in renal interstitial fibroblasts.

Iron is an essential mineral for oxygen delivery and for a variety of enzymatic activities, but excessive iron results in oxidative cytotoxicity. Because iron is primarily used in red blood cells, defective erythropoiesis caused by loss of the erythroid growth factor erythropoietin (Epo) elevates iron storage levels in serum and tissues. Here, we investigated the effects of iron in a mouse model of Epo-deficiency anemia, in which serum iron concentration was significantly elevated. We found that intraperitoneal injection of iron-dextran caused severe iron deposition in renal interstitial fibroblasts, the site of Epo production. Iron overload induced by either intraperitoneal injection or feeding decreased activity of endogenous Epo gene expression by reducing levels of hypoxia-inducible transcription factor 2α (HIF2α), the major transcriptional activator of the Epo gene. Administration of an iron-deficient diet to the anemic mice reduced serum iron to normal concentration and enhanced the ability of renal Epo production. These results demonstrate that iron overload due to Epo deficiency attenuates endogenous Epo gene expression in the kidneys. Thus, iron suppresses Epo production by reducing HIF2α concentration in renal interstitial fibroblasts.

Epoetin beta pegol for treatment of anemia ameliorates deterioration of erythrocyte quality associated with chronic kidney disease.

BACKGROUND: Epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.) is currently widely used for the treatment of anemia associated with chronic kidney disease (CKD). Therapeutic control of anemia is assessed by monitoring haemoglobin (Hb) levels. However, certain qualitative aspects of erythrocytes are also impaired in CKD, including loss of deformability and shortened life-span. Therefore, monitoring Hb alone could potentially fail to reveal pathological changes in erythrocytes. Focusing on erythrocyte quality in CKD may lead to more effective anemia therapy with C.E.R.A. METHODS: A CKD rat model was induced by uninephrectomy followed by anti-Thy1.1 antibody injection. From 5 weeks after the operation, C.E.R.A. (0.6 µg/kg) or vehicle was administered every 2 weeks. Erythrocyte deformability was quantified with ektacytometry and erythrocyte turnover was estimated by biotin labeling. Intracellular calcium level was assessed by Fluo-3/AM. RESULTS: Erythrocyte deformability progressively declined in CKD rats. Furthermore, erythrocyte turnover in the circulation drastically accelerated in CKD rats. With administration of C.E.R.A. at a dose sufficient to adequately control Hb, deterioration of erythrocyte deformability and turnover in CKD rats were significantly improved. Intracellular calcium, which plays a pivotal role in the mediation of erythrocyte quality, was significantly increased in CKD and was normalized by C.E.R.A. CONCLUSION: C.E.R.A. treatment exerted a favorable effect not only on anemia but also on the improvement of erythrocyte quality. C.E.R.A. administered for the treatment of CKD-associated anemia may confer therapeutic benefits on erythrocytes.

Visualization of 57Fe-Labeled Heme Isotopic Fine Structure and Localization of Regions of Erythroblast Maturation in Mouse Spleen by MALDI FTICR-MS Imaging.

Epoetin beta pegol (continuous erythropoiesis receptor activator; C.E.R.A.), or methoxy-polyethylene glycol-modified epoetin beta, is a long-acting erythropoiesis stimulating agent (ESA) that effectively maintains hemoglobin levels. It promotes proliferation of erythroid progenitor cells in hematopoietic organs and leads to increased reticulocyte and hemoglobin levels. However, the detailed erythropoietic effects of various ESAs on their target organs have yet to be clarified, and new approaches are needed to analyze tissue iron localization with structural information. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) techniques are widely used in basic pharmaceutical research. High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) imaging enables the spatial mapping and identification of biomolecules. In this study, mice administered with C.E.R.A. were fed a diet containing the stable iron isotope 57Fe. The 57Fe-heme+ isotopic fine structure peak (m/z 617.1772) was separated from the non-labeled heme+ isotopic peak (Δ0.0029) by FTICR-MS with a resolving power of more than 500,000. We optimized the platform to analyze the distribution of 57Fe-heme in the spleen using MALDI FTICR-MS imaging. The combination of the ultrahigh resolution power of FTICR-MS and a stable isotope labeling technique has the potential to be very effective in basic pharmaceutical research. Graphical Abstract ᅟ.

The effect of frequency of C.E.R.A. administration on the contribution of dietary iron for erythropoiesis.

Erythropoiesis-stimulating agents are among the therapeutic options for renal anemia. Under erythropoietic stimulation, the synthesis of hemoglobin requires a large amount of iron, which is supplied both from absorption of dietary iron and the mobilization of stored iron. However, under iron-loading conditions, dietary iron absorption is suppressed via down-regulation of duodenal iron transporters. Because the contribution of dietary iron is essential for erythropoiesis, we aimed to investigate the conditions in which dietary iron is efficiently used for erythropoiesis. To quantify the contribution of dietary iron for erythropoiesis, we labelled dietary iron using the stable iron isotope 57Fe, and assessed 57Fe-derived hemoglobin synthesis under erythropoietic stimulation by C.E.R.A. (Continuous Erythropoietin Receptor Activator) and iron loading in mice. Our results show that the contribution of dietary iron to erythropoiesis is not changed by different increments of C.E.R.A. dose. However, iron loading suppressed the contribution of dietary iron to erythropoiesis. To confirm these results under conditions mimicking clinical settings, we compared single and intermittent administration of C.E.R.A. in mice under both physiological and iron-loaded conditions, and found that the contribution of dietary iron to erythropoiesis is more strongly affected by body iron status than by erythropoietic stimulation.

Epoetin beta pegol ameliorates flow-mediated dilation with improving endothelial nitric oxide synthase coupling state in nonobese diabetic rats.

BACKGROUND/AIMS: Patients with diabetic nephropathy have a high cardiovascular mortality. Epoetin beta pegol (continuous erythropoietin receptor activator, C.E.R.A.) is a drug for the treatment of renal anemia. In this study, we investigated the effect of C.E.R.A. on vascular endothelial function as evaluated by flow-mediated dilation (FMD) and the relationship between hematopoiesis and FMD in diabetic nephropathy rats. METHODS: Male Spontaneously Diabetic Torii rats (SDT, 22 weeks old) were used. C.E.R.A. (0.6, 1.2 µg/kg) was administered subcutaneously once every 2 weeks for 8 weeks. At 1 week after last administration (31 weeks old), we assessed FMD in the femoral arteries of anesthetized rats using a high-resolution ultrasound system. FMD was also measured 1 week after single C.E.R.A. treatment (5.0 µg/kg) to examine the influence of hematopoiesis. RESULTS: Flow-mediated dilation was significantly decreased in SDT rats before the start of C.E.R.A. treatment (22 weeks old). Repeated administration of C.E.R.A. dose-dependently improved FMD in SDT rats (31 weeks old) without changing blood glucose, nitroglycerin-induced vasodilation, or kidney function. Long-term administration of C.E.R.A. improved the state of endothelial nitric oxide synthase uncoupling in the femoral arteries of SDT rats, which showed a positive correlation with FMD. On the other hand, there was no correlation between FMD and Hb or Hct in SDT rats. Furthermore, at 1 week after single administration of C.E.R.A., FMD was not significantly improved although hemoglobin levels were comparable with levels following long-term C.E.R.A. CONCLUSION: Long-term treatment with C.E.R.A. improved FMD in SDT rats even after onset of endothelial dysfunction.

Treatment with anti-IL-6 receptor antibody prevented increase in serum hepcidin levels and improved anemia in mice inoculated with IL-6-producing lung carcinoma cells.

BACKGROUND: Hepcidin, a key regulator of iron metabolism, is produced mainly by interleukin-6 (IL-6) during inflammation. A mechanism linking cancer-related anemia and IL-6 through hepcidin production is suggested. To clarify the hypothesis that overproduction of IL-6 elevates hepcidin levels and contributes to the development of cancer-related anemia, we evaluated anti-IL-6 receptor antibody treatment of cancer-related anemia in an IL-6-producing human lung cancer xenograft model. METHODS: Nude mice were subcutaneously inoculated with cells of the IL-6-producing human lung cancer cell line LC-06-JCK and assessed as a model of cancer-related anemia. Mice bearing LC-06-JCK were administered rat anti-mouse IL-6 receptor antibody MR16-1 and their serum hepcidin levels and hematological parameters were determined. RESULTS: LC-06-JCK-bearing mice developed anemia according to the production of human IL-6 from xenografts, with decreased values of hemoglobin, hematocrit, and mean corpuscular volume (MCV) compared to non-tumor-bearing (NTB) mice. LC-06-JCK-bearing mice showed decreased body weight and serum albumin with increased serum amyloid A. MR16-1 treatment showed significant inhibition of decreased body weight and serum albumin levels, and suppressed serum amyloid A level. There was no difference in tumor volume between MR16-1-treated mice and immunoglobulin G (IgG)-treated control mice. Decreased hemoglobin, hematocrit, and MCV in LC-06-JCK-bearing mice was significantly relieved by MR16-1 treatment. LC-06-JCK-bearing mice showed high red blood cell counts and erythropoietin levels as compared to NTB mice, whereas MR16-1 treatment did not affect their levels. Serum hepcidin and ferritin levels were statistically elevated in mice bearing LC-06-JCK. LC-06-JCK-bearing mice showed lower values of MCV, mean corpuscular hemoglobin (MCH), and serum iron as compared to NTB mice. Administration of MR16-1 to mice bearing LC-06-JCK significantly suppressed levels of both serum hepcidin and ferritin, with increased values of MCV and MCH. CONCLUSIONS: Our results suggest that overproduction of hepcidin by IL-6 signaling might be a major factor that leads to functionally iron-deficient cancer-related anemia in the LC-06-JCK model. We demonstrated that inhibition of the IL-6 signaling pathway by MR16-1 treatment resulted in significant recovery of iron-deficiency anemia and alleviation of cancer-related symptoms. These results indicate that IL-6 signaling might be one possible target pathway to treat cancer-related anemia disorders.

Epoetin beta pegol, but not recombinant erythropoietin, retains its hematopoietic effect in vivo in the presence of the sialic acid-metabolizing enzyme sialidase.

Erythropoiesis-stimulating agents (ESAs) are widely used for treating chronic kidney disease (CKD)-associated anemia. The biological activity of ESAs is mainly regulated by the number of sialic acid-containing carbohydrates on the erythropoietin (EPO) peptide. Sialidase, a sialic acid-metabolizing enzyme that accumulates in CKD patients, is suspected of contributing to shortening the circulation half-life of ESAs. Epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), is an EPO integrated with methoxypolyethylene glycol (PEG). It has been suggested that C.E.R.A. may exert a favorable therapeutic effect, even under conditions of elevated sialidase; however, no detailed investigation of the pharmacological profile of C.E.R.A. in the presence of sialidase has been reported. In the present study, we injected C.E.R.A. or EPO pre-incubated with sialidase into rats, and assessed the hematopoietic effect by reticulocyte count. The hematopoietic effect of C.E.R.A., but not EPO, was preserved after sialidase treatment, despite the removal of sialic acid. Proliferation of EPO-dependent leukemia cells (AS-E2) was significantly increased by desialylated C.E.R.A. and EPO compared to non-treated C.E.R.A. or EPO. In conclusion, we show that C.E.R.A. exerts a favorable hematopoietic effect even under conditions of elevated sialidase. Our findings may contribute to a better understanding of CKD and more effective therapeutic approaches based on a patient's profile of anemia.

Quantitative analysis of dietary iron utilization for erythropoiesis in response to body iron status.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis. There are two sources of iron for erythropoiesis, dietary and stored iron; however, their relative contributions to erythropoiesis remain unknown. In this study, we used the stable iron isotope (57)Fe to quantify synthesis of hemoglobin derived from dietary iron. Using this method, we investigated the activities of dietary iron absorption and the utilization of dietary iron for erythropoiesis in responses to stimulated erythropoiesis and to interventions to alter body iron status. Under iron-loaded conditions, the activity of dietary iron absorption was clearly lowered in response to up-regulation of hepcidin, although the estimated activity of iron release from stored iron was not compared with that under control conditions. This result was supported by the observation that two duodenal iron transporters, divalent metal transporter 1 (DMT1) and ferroportin, were downregulated by iron loading, although the levels of expression of ferroportin in iron storage tissues were not changed by iron loading under erythropoietic stimulation by epoetin-ß pegol (C.E.R.A., a long-acting erythropoiesis-stimulating agent). These results indicate that the dietary iron absorption system is more sensitive to body iron status than are reticuloendothelial iron- release mechanisms. Our data indicated that there could be a regulatory mechanism favoring use of stored iron over dietary iron under iron-loaded conditions.

Reduction of a marker of oxidative stress with enhancement of iron utilization by erythropoiesis activation following epoetin beta pegol administration in iron-loaded db/db mice.

UNLABELLED: Iron, an essential element for various biological processes, can induce oxidative stress. We hypothesized that iron utilization for erythropoiesis, stimulated by epoetin beta pegol (C.E.R.A.), a long-acting erythropoiesis-stimulating agent, contributes to the reduction of iron-induced oxidative stress. We first investigated the sensitivity of several biomarkers to detect oxidative stress in mice by altering the amount of total body iron; we then investigated whether C.E.R.A. ameliorated oxidative stress through enhanced iron utilization. We treated db/db mice with intravenous iron-dextran and evaluated several biomarkers of iron-induced oxidative stress. In mice loaded with 5 mg/head iron, hepatic iron content was elevated and the oxidative stress marker d-ROMs (serum derivatives of reactive oxygen metabolites) was increased, whereas urinary 8-hydroxy-2'-deoxyguanosine and serum malondialdehyde were not, indicating that d-ROMs is a sensitive marker of iron-induced oxidative stress. To investigate whether C.E.R.A. ameliorated oxidative stress, db/db mice were intravenously administered iron-dextran or dextran only, followed by C.E.R.A. Hemoglobin level increased, while hepatic iron content decreased after C.E.R.A. TREATMENT: Serum d-ROMs decreased after C.E.R.A. treatment in the iron-dextran-treated group. Our results suggest that C.E.R.A. promotes iron utilization for erythropoiesis through mobilization of hepatic iron storage, leading to a decrease in serum oxidative stress markers in iron-loaded db/db mice.

The three isoforms of hepcidin in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS).

Hepcidin, the iron regulatory hormone, has three isoforms; -20, -22 and -25. While hepcidin-25 has been studied extensively, the physiological significance of other isoforms remains poorly understood. Using a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-tandem MS) developed by our group, we quantified hepcidin isoforms in human serum to elucidate their characteristics, and investigated the role of hepatocytes in isoform processing. Hepcidin isoforms in serum obtained from 40 healthy volunteers were quantified. Synthetic hepcidin peptides were added to healthy serum, and to HepG2 culture media, and hepcidin isoform concentrations determined. All three hepcidin isoforms were detected in human serum; however, hepcidin-25 concentrations were highest. The three hepcidin isoforms showed a strong positive correlation with each other and with serum ferritin. Additionally, while hepcidin-20 was strongly correlated with serum creatinine, the other isoforms were not. Hepcidin-20 and -25 levels were also increased in chronic kidney disease (CKD) serum. Hepcidin-22 rapidly degraded into hepcidin-20, whereas hepcidin-25 remained relatively stable. Finally, hepcidin-22 degradation into hepcidin-20 was accelerated in the presence of HepG2. This method has enabled us to reveal fundamental characteristics of the three hepcidin isoforms in serum and may be a powerful tool for quantifying hepcidin isoform expression and processing.

Epoetin beta pegol alleviates oxidative stress and exacerbation of renal damage from iron deposition, thereby delaying CKD progression in progressive glomerulonephritis rats.

The increased deposition of iron in the kidneys that occurs with glomerulopathy hinders the functional and structural recovery of the tubules and promotes progression of chronic kidney disease (CKD). Here, we evaluated whether epoetin beta pegol (continuous erythropoietin receptor activator: CERA), which has a long half-life in blood and strongly suppresses hepcidin-25, exerts renoprotection in a rat model of chronic progressive glomerulonephritis (cGN). cGN rats showed elevated urinary total protein excretion (uTP) and plasma urea nitrogen (UN) from day 14 after the induction of kidney disease (day 0) and finally declined into end-stage kidney disease (ESKD), showing reduced creatinine clearance with glomerulosclerosis, tubular dilation, and tubulointerstitial fibrosis. A single dose of CERA given on day 1, but not on day 16, alleviated increasing uTP and UN, thereby delaying ESKD. In the initial disease phase, CERA significantly suppressed urinary 8-OHdG and liver-type fatty acid-binding protein (L-FABP), a tubular damage marker. CERA also inhibited elevated plasma hepcidin-25 levels and alleviated subsequent iron accumulation in kidneys in association with elevated urinary iron excretion and resulted in alleviation of growth of Ki67-positive tubular and glomerular cells. In addition, at day 28 when the exacerbation of uTP occurs, a significant correlation was observed between iron deposition in the kidney and urinary L-FABP. In our study, CERA mitigated increasing kidney damage, thereby delaying CKD progression in this glomerulonephritis rat model. Alleviation by CERA of the exacerbation of kidney damage could be attributable to mitigation of tubular damage that might occur with lowered iron deposition in tubules.

Treatment with the combination of ibandronate plus eldecalcitol has a synergistic effect on inhibition of bone resorption without suppressing bone formation in ovariectomized rats.

Bisphosphonates are widely used in the treatment of osteoporosis and contribute to the reduction of bone fractures. Ibandronate (IBN) is a highly potent, nitrogen-containing bisphosphonate, which is administered orally or intravenously at extended dosing intervals. Vitamin D or active vitamin D3 derivatives are also used in the treatment of osteoporosis, and are often used in combination with other drugs. In this study, we investigated the effect of treatment with the combination of once-monthly s.c. dosing of IBN plus once-daily oral eldecalcitol (ELD), an active vitamin D3 derivative, using aged ovariectomized (OVX) rats. Treatment was started the day after OVX, and analyses were performed 4, 8, and 12 weeks thereafter by determination of bone markers, bone mineral density, biomechanical properties, and histomorphometry. The combination treatment showed a synergistic effect in increasing both lumbar and femoral BMD, and resulted in a significant increase in bone ultimate load. The combination of IBN plus ELD acted synergistically to reduce bone resorption, whereas bone formation did not decrease any more than with monotherapy with either IBN or ELD. Bone formation independent of bone resorption (a process known as 'minimodeling') was not changed in vehicle treated OVX rats despite the increase in bone turnover. ELD upregulated minimodeling, which was however not diminished in the combination treatment. In conclusion, treatment with the combination of IBN plus ELD was beneficial in the treatment of osteoporosis in aged OVX rats. It exhibited a synergistic inhibitory effect on bone resorption and keeps bone formation at the level of sham controls. This uncoupling of bone resorption/bone formation was affected, to some extent, by minimodeling-based bone formation which is independent of bone resorption. This combination regimen which showed synergistic effect on BMD and bone ultimate load without inhibition of bone formation may be beneficial in long-term osteoporosis treatment to prevent bone fractures.

Hepcidin/ferroportin expression levels involve efficacy of pegylated-interferon plus ribavirin in hepatitis C virus-infected liver.

AIM: To investigate the relationship between the iron-metabolism-related gene expression profiles and efficacy of antiviral therapy in chronic hepatitis C patients. METHODS: The hepatic expression profile of iron-metabolism-related genes was analyzed and its association with virological response to pegylated-interferon plus ribavirin combination therapy was evaluated. A hundred patients with chronic hepatitis C (genotype1b, n = 50; genotype 2, n = 50) were enrolled and retrospectively analyzed. Liver biopsy samples were subjected to quantitative polymerase chain reaction for iron-metabolism-related genes and protein expression (Western blotting analysis) for ferroportin. As a control, normal liver tissue was obtained from 18 living donors of liver transplantation. Serum hepcidin level was measured by sensitive liquid chromatography/electrospray ionization tandem mass spectrometry. RESULTS: Iron overload is associated with liver damage by increasing oxidative stress and hepatitis C virus (HCV) is reported to induce iron accumulation in hepatocytes in vivo. Conversely, iron administration suppresses HCV replication in vitro. Therefore, the association between HCV infection and iron metabolism remains unclear. Compared with controls, patients had significantly higher gene expression for transferrin, iron-regulatory proteins 1 and 2, divalent metal transporter 1, and ferroportin, but similar for transferrin receptors 1 and 2, and hepcidin. When the expression profiles were compared between sustained virological response (SVR) and non-SVR patients, the former showed significantly lower transcription and protein expression of hepcidin and ferroportin. Expression of hepcidin-regulating genes, BMPR1, BMPR2, and hemojuvelin, was significantly increased, whereas BMP2 was decreased in HCV-infected liver. BMPR2 and hemojuvelin expression was significantly lower in the SVR than non-SVR group. HCV infection affects the expression of iron-metabolism-related genes, leading to iron accumulation in hepatocytes. CONCLUSION: Decreased expression of hepcidin and ferroportin in SVR patients indicates the importance of hepatocytic iron retention for viral response during pegylated-interferon plus ribavirin treatment.

Branched-chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein-expressing mice.

BACKGROUND & AIMS: Branched-chain amino acids (BCAA) reduce the incidence of hepatocellular carcinoma (HCC) in patients with cirrhosis. However, the mechanisms that underlie these effects remain unknown. Previously, we reported that oxidative stress in male transgenic mice that expressed hepatitis C virus polyprotein (HCVTgM) caused hepatic iron accumulation by reducing hepcidin transcription, thereby leading to HCC development. This study investigated whether long-term treatment with BCAA reduced hepatic iron accumulation and oxidative stress in iron-overloaded HCVTgM and in patients with HCV-related advanced fibrosis. METHODS: Male HCVTgM were fed an excess-iron diet that comprised either casein or 3.0% BCAA, or a control diet, for 6 months. RESULTS: For HCVTgM, BCAA supplementation increased the serum hepcidin-25 levels and antioxidant status [ratio of biological antioxidant potential (BAP) relative to derivatives of reactive oxygen metabolites (dROM)], decreased the hepatic iron contents, attenuated reactive oxygen species generation, and restored mitochondrial superoxide dismutase expression and mitochondrial complex I activity in the liver compared with mice fed the control diet. After 48 weeks of BCAA supplementation in patients with HCV-related advanced fibrosis, BAP/dROM and serum hepcidin-25 increased and serum ferritin decreased compared with the pretreatment levels. CONCLUSIONS: BCAA supplementation reduced oxidative stress by restoring mitochondrial function and improved iron metabolism by increasing hepcidin-25 in both iron-overloaded HCVTgM and patients with HCV-related advanced fibrosis. These activities of BCAA may partially account for their inhibitory effects on HCC development in cirrhosis patients.

Epoetin beta pegol (C.E.R.A.) promotes utilization of iron for erythropoiesis through intensive suppression of serum hepcidin levels in mice.

Epoetin beta pegol (C.E.R.A.) is a novel, long-acting, erythropoiesis-stimulating agent. We investigated iron utilization associated with erythropoiesis, and how it is affected by the long-term suppression of hepcidin following C.E.R.A. treatment in mice. C57BL/6N mice were administered a single intravenous injection of C.E.R.A. Hemoglobin (Hb) levels were significantly elevated following C.E.R.A. administration, while serum hepcidin levels were continuously suppressed. Expression levels of duodenal iron transporters, ferroportin (FPN) which is internalized by hepcidin binding and divalent metal transporter 1 (DMT1), increased after C.E.R.A. administration. To evaluate the contribution of dietary iron absorption to erythropoiesis following C.E.R.A. treatment, C.E.R.A.-treated mice were fed either an iron-deficient diet to restrict dietary iron absorption or a control diet. The restriction of dietary iron absorption resulted in impaired Hb elevation after C.E.R.A. administration. To investigate the utilization of stored iron for erythropoiesis following C.E.R.A. treatment, iron indices were analyzed in C.E.R.A.-treated mice given an intraperitoneal pre-treatment injection of iron-dextran. Serum hepcidin levels and splenic hemosiderin deposition decreased after C.E.R.A. administration. These results indicate that C.E.R.A. promotes utilization of iron for erythropoiesis both through enhancement of dietary iron absorption and mobilization of iron storage from reticuloendothelial cells.

Hepcidin production in response to iron is controlled by monocyte-derived humoral factors.

Hepcidin, which is mainly produced by the liver, is the key regulator in iron homeostasis. Hepcidin expression is up-regulated by iron loading in vivo, but the mechanism underlying this process is not completely understood. In the present study, we investigated the mechanism, following the hypothesis that hepcidin production in response to iron loading is regulated by extra-hepatic iron sensors. We measured serum hepcidin concentrations and iron indices in Wistar rats treated with saccharated ferric oxide (SFO). Human hepatoma-derived HepG2 cells were stimulated using SFO-administered rat sera, and co-cultured with rat spleen cells, human monocyte-derived THP-1 cells, or human monocytes with diferric transferrin (holo-Tf), and hepcidin concentrations in the conditioned media were measured. SFO elevated rat serum hepcidin concentrations. SFO-treated rat sera increased hepcidin production from HepG2 cells, and this induction correlated with serum hepcidin levels, but not with iron indices. Holo-Tf up-regulated hepcidin concentrations in media from HepG2 cells co-cultured with rat spleen cells, THP-1 cells, or human monocytes with or without cell-to-cell contacts, while holo-Tf did not up-regulate hepcidin from HepG2 cells alone. Our results suggest the existence of humoral factors capable of inducing hepcidin production that are secreted by extra-hepatic cells, such as reticuloendothelial monocytes, in response to iron.

Upregulation of iron regulatory hormone hepcidin by interferon α.

BACKGROUND AND AIM: Interferon (IFN) activates various immune systems in vivo and is administered to patients with diseases such as viral hepatitis B, C, and malignant tumors. Iron dysregulation has been reported during treatment with IFN; however, it remains unclear whether IFN itself affects iron metabolism. We therefore determined the effect of IFN on iron metabolism. METHODS: Mouse IFNα was administered to mice, and serum, spleen, bone marrow, liver, and duodenum tissue samples were subsequently collected. The messenger RNA (mRNA) and protein expression of genes involved in iron metabolism were then analyzed by real-time reverse transcription-polymerase chain reaction, Western blotting, and liquid chromatography-tandem mass spectrometry. Immunofluorescence for ferroportin was also performed. RESULTS: Among the gene expressions analyzed, we found that the expression of hepcidin, an iron regulatory hormone produced in the liver, was highly upregulated after IFNα treatment. Serum hepcidin levels and hepcidin mRNA expression in the liver were both found to be increased in the IFNα-treated mice. The expression of ferroportin (the target molecule of hepcidin) in the duodenum of the IFNα-treated mice was observed to be decreased, indicating that hepcidin upregulation could be physiologically functional. In vitro analysis of primary hepatocytes treated with IFNα and human hepatoma-derived cells showed an upregulation of hepcidin mRNA, including an activation of signal transducer and activator of transcription3, which was shown to be involved in the hepcidin upregulation. CONCLUSIONS: Results indicate that iron absorption is decreased during IFN treatment; this favorable effect could inhibit iron overload during IFN treatment and may enhance the action of IFN.

The removal of serum hepcidin by different dialysis membranes.

PURPOSE: Hepcidin has been suspected to be associated with anemia of chronic disease, which is commonly observed in patients with maintenance hemodialysis (MHD). As almost of hepcidin is bounded to protein, it is essential to clarify which kind of dialysis membrane can remove it efficiently. METHODS: Ex vivo study: 50 mL of whole blood from healthy volunteers were circulated for 2 h in a microcircuit with mini-dialyzers (acrylonitrile-co-methallyl sulfonate (AN69) or polysulfone (PS)) without ultrafiltration. We measured hepcidin-25 levels at 0, 60, and 120 min in the blood samples. In vivo study: Blood samples were taken from 28 MHD patients at the start and end of HD sessions with PS or AN69. We measured serum levels of hepcidin 20, 22, and 25 by liquid chromatography tandem mass spectrometry, and also measured serum levels of urea nitrogen (UN), ß2microglobulin (MG). RESULTS: Ex vivo study: Although serum hepcidin 25 levels increased after the ex vivo session with PS, they significantly decreased with AN69. In vivo study: The reduction ratio of ß2MG by PS was significantly higher than that of AN69. On the other hand, there was no significant difference in the reduction ratio of hepcidin 20, 22, and 25 between PS and AN69. CONCLUSIONS: Both super-flux PS and AN69 similarly removed hepcidin 20 22, and 25. HD with PS might achieve a high removal ratio of hepcidin by enhanced diffusion performance and an increased clearance of small molecule solutes. On the other hand, AN69 might remove hepcidin by adsorption.

Erythropoietin stimulation decreases hepcidin expression through hematopoietic activity on bone marrow cells in mice.

Erythropoiesis-stimulating agents (ESA) are now central to the treatment of renal anemia and are associated with improved clinical outcomes. It is well known that erythropoietin (EPO) is a key regulator of erythropoiesis through its promotion of red blood cell production. In order to investigate the role of ESA on iron metabolism, we analyzed the regulation of the iron regulatory hormone hepcidin by ESA treatment in a bone marrow transplant model in mouse. After treating C57BL/6 mice with continuous erythropoietin receptor activator (C.E.R.A.), recombinant human epoetin-ß (rhEPO), or recombinant human carbamylated epoetin-ß (rhCEPO), we investigated serum hepcidin concentrations and parameters of erythropoiesis. Serum hepcidin concentrations after rhEPO treatment were analyzed in mice subjected to total body irradiation followed by bone marrow transplantation. C.E.R.A. administration caused long-term downregulation of serum hepcidin levels. Serum hepcidin levels in rhEPO-treated mice decreased significantly, whereas there was no change in rhCEPO-treated mice. The reduction in circulating hepcidin levels after rhEPO administration was not observed in irradiated mice. Finally, bone marrow transplantation recovered the response to rhEPO administration that downregulates hepcidin concentration in irradiated mice. These results indicate that ESA treatment downregulates serum hepcidin concentrations, mainly by indirect mechanisms affecting hematopoietic activity in bone marrow cells.